Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy

Abstract

Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.

FIG. 1.

Summary of CRISPR-mediated approaches to treat DMD mutations. (a) CRISPR-DMD approaches mainly target dystrophin to precisely correct specific mutation(s) including (1) Cas9 dual DSBs to excise exons restoring reading frame or single DSBs knocking out splice sites, (2) ABE substitutions mutating splice sites, and (3) prime-editing mutating splice sites or deleting and adding integrase recognition sites for future replacement. (b) Alternative approaches target utrophin, an autosomal homolog of dystrophin, to upregulate its expression supplanting dystrophin.

FIG. 2.

Summary of the delivery vectors that have been used in CRISPR-DMD research. Physical and non-viral vector delivery are mostly applied in in vitro stage, while viral vectors are heavily used in preclinical work in mice and dog models of DMD.

FIG. 3.

Schematic figure of cellular and humoral immune response against CRISPR therapeutic delivery. (a) Immune responses targeting LNPs include neutralizing preexisting antibodies against PEG and other LNP surface components, which result in APC endocytosis clearing the LNPs and inciting an immune response. Cytotoxic T cells can recognize LNPs and secrete inflammatory cytokines. Endosomal LNP recognition and MHC II presentation can result in mounting an inflammatory response and degradation of internal components can occur after nuclear entry. (b) Immune responses targeting AAV include neutralizing pre-existing antibodies against the AAV capsid, resulting in APC endocytosis clearing the AAV and inciting an immune response. Cytotoxic T cells can recognize AAV and secrete inflammatory cytokines. Endosomal recognition of AAV and MHC II presentation of AAV capsid proteins to T cells and B cells will also mount an immune response. Degradation of internal components can occur after nuclear entry. (c) Preexisting antibodies against Cas9 have been detected, neutralizing, and clearing the protein.